All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Phosphatidylserine (PS), a phospholipid nutrient, is active in cell membranes and is the major acidic phospholipid component in the membrane of brain cells. PS plays a crucial role in many membrane-associated nerve cell processes. The main purpose of PS is to help maintain proper membrane fluidity, which has major implications on most membrane functions.
PS has been the subject of numerous human clinical trials of memory loss, mood, cognitive performance and learning ability. Many of the studies show that PS can be helpful for those with age-related memory impairment. Moreover, PS can even help to optimize cognition in those with no cognitive impairment.
Dietary PS is efficiently and rapidly absorbed in the intestine, taken up into the blood, and readily crosses the blood-brain barrier to reach the nerve cells in the brain.
PS can be extracted from bovine brain, from plants or it can be produced from soybean lecithin using biocatalysis. By using the transphosphatidylation reaction with phospholipases D (PLDs), the head group of phospholipids can be modified easily. Thus, phosphatidylserine can be produced from phosphatidylcholine or any other phospholipid mixture with serine through PLD catalysis.
Currently, PS is manufactured and marketed in powder and fluid forms, at different concentrations, ranging from 10% to 90%. The fluid form of the PS commonly consists of a clear and transparent solution of phosphatidylserine, usually in oily media of medium-chain triglycerides (MCT) or soy triglycerides. This form is commonly used for dietary supplements in the form of softgel capsules. PS supplements fall within the category of nutraceuticals, which are defined as any substance that is a food, or part of a food and provides medical and/or health benefits, including the prevention and treatment of disease. In the broad definition, both dietary supplements and functional foods are considered nutraceuticals.
One of the main difficulties in phosphatidylserine preparations, especially in liquid form, is its low stability due to rapid decomposition. The exact cause of this decomposition is not fully understood. There are many hypotheses regarding the cause of this phenomenon, although most are not scientifically established or proved. The common belief is that decomposition is caused mainly by residual biocatalytic activity and/or side-reactions with water or glycerol as well as other alcohol moieties. These reactions can be especially important when the PS preparation is fluid, and it is encapsulated in softgel capsules. Softgel encapsulation usually results in the migration and subsequent incorporation of low levels of water and/or glycerol into the capsule content.
PS preparations, and especially fluid preparations, in which PLD residual biocatalytic activity is present may be susceptible to PS biocatalytic degradation by transphosphatidylation, which removes the serine head group, resulting in loss of the PS active ingredient. This transphosphatidylation activity can result in hydrolysis utilizing water found in the fluid PS preparation itself or the fluid preparation following encapsulation. Hydrolysis will lead to the formation of phosphatidic acid (PA). In case the transphosphatidylation utilizes glycerol or other alcohol moieties found in the fluid PS preparation itself or the fluid preparation following encapsulation, this may lead to the replacement of the serine head group with other alcohols, yielding phosphatidylglycerol (PG) or other corresponding phospholipid derivatives.
Other degradation routes are also possible. These include chemical degradation, like for example decarboxylation of the serine carboxylic group, yielding products such as phosphatidylethanolamine (PE) or other derivatives. Lipid peroxidation may also play a role in PS degradation. PS can be degraded by full or partial hydrolysis of the phospholipid fatty acids, yielding de-acylated PS (GPS) or lyso-PS (LPS), correspondingly. In case of PS phosphate removal, either enzymatically by enzymes with phospholipase C (PLC)-like activity or chemically, diglycerides can be created, also resulting in reduction of the PS active ingredient.
One way to overcome degradation has been proposed in WO 03/088949, wherein the phospholipid is embedded in a hard or paste-like matrix.
Besides what has been suggested above, other degradation pathways are plausible and might be responsible to the apparent degradation of PS in commercial preparations.
It is therefore an object of the present invention to provide stabilized PS preparations, as powders, liquids or dispersions.
It is a further object of the present invention to provide methods for the preparation of such stabilized PS preparations.
It is yet a further object of the present invention to provide the said stabilized preparations for use in common dietary supplements applications, and particularly in softgel capsules.
It is yet a further object of the present invention to provide the said stabilized preparations for use as stand-alone nutraceuticals or as additives to food articles or to pharmaceutical compositions.
These and other objects of the invention will become apparent as the description proceeds.